CN109481472A - Application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment inflammatory bowel disease - Google Patents

Application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment inflammatory bowel disease Download PDF

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Publication number
CN109481472A
CN109481472A CN201710812667.5A CN201710812667A CN109481472A CN 109481472 A CN109481472 A CN 109481472A CN 201710812667 A CN201710812667 A CN 201710812667A CN 109481472 A CN109481472 A CN 109481472A
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bacteroides fragilis
capsular polysaccharide
extract
sediment
group
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智发朝
王晔
刘洋洋
白杨
王从峰
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Guangzhou Zhiyi Biotechnology Co Ltd
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Guangzhou Zhiyi Biotechnology Co Ltd
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Priority to CN201710812667.5A priority Critical patent/CN109481472A/en
Priority to PCT/CN2017/120113 priority patent/WO2019047440A1/en
Publication of CN109481472A publication Critical patent/CN109481472A/en
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    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Abstract

The present invention relates to application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment inflammatory bowel disease, which is bacteroides fragilis capsular polysaccharide A.The bacteroides fragilis capsular polysaccharide A that molecular weight is 5-70KD has unexpectedly been prepared in the present invention, and it is surprised to find that molecular weight is that the capsular polysaccharide A of 5~70KD has the function of preferably preventing and treating inflammatory bowel disease, effect is much better than the capsular polysaccharide A that the molecular weight extracted from bacteroides fragilis NCTC 9343 is 110KD.

Description

Bacteroides fragilis extract is in the drug or food of preparation prevention and treatment inflammatory bowel disease Using
Technical field
The present invention relates to the applied technical fields of bacteroides fragilis, are making more particularly to a kind of bacteroides fragilis extract The drug of standby prevention and treatment inflammatory bowel disease or the application in food.
Background technique
Inflammatory bowel disease (Inflammatorybowel disease, IBD) is the chronic gut inflammation of one group of unknown cause Property disease, including ulcerative colitis (Ulcerative colitis, UC) and Crohn disease (Crohn's disease, CD). The former is also known as nonspecific ulcerative colitis, is the inflammation of a kind of rectum that reason is unknown and colon, and lesion is limited primarily to greatly Intestinal mucosa and submucosa.The latter is a kind of chronic granulomatous inflammation, and lesion can be involved each position of gastrointestinal tract, and be returned with latter end It intestines and closes on based on colon, is in segmental, the investigation on asymmetric distribution (understanding and therapeutic advance of Jiang Weiru inflammatory bowel disease more The Shanghai [J] medicine, 2010,05:207-210.).The pathogenesis of inflammatory bowel disease not yet illustrates, and is presently considered to be on gene Susceptible population generates caused by excessive congenital or acquired immunity reaction (Jiang Weiru inflammatory bowel disease to commensal gut microorganism Understanding and the Shanghai therapeutic advance [J] medicine, 2010,05:207-210.).
Western countries' IBD age of onset is in bimodal shape distribution more, and the first age of onset peak UC is 30-39 years old, and CD is 20-29 years old, it was more with first peak case load that the second age of onset peak UC and CD, which is 60-70 years old,.Asian countries IBD The second peak of age of onset is rare, UC and CD age of onset peak postpones 10 years compared with western countries.
In the past 20 years, IBD case load rapidly increases at home.1989-2007 Nian Jian China IBD document report case load by Cumulative more (Hu RW, Ouyang Q, ChenX et al.Analysis ofthe articles of inflammatorybowel disease inthe literature Chinainrecent fifteenyears[J].Chin J Gastroenterol, 2007,12:74-77.).(Jiang XL, the Cui HF.An analysis of such as Jiang 10218ulcerative colitis cases in China [J] .World J Gastroenterol, 2002,8 (1): 10218 UC patients of 1981-2000 domestic literature report 158-161.) are analyzed, case load rises between discovery 10 years 3.08 again.(WangY, the Ouyang Q such as Wang；APDW2004Chinese IBD working group.Ulcerative colitis in China:retrospective analysis of 3100hospitalizedpatients[J].J Gastroenterol Hepatol, 2007,22 (9): 1450-1455.) and IBD working group, China (APDW 2004Chinese IBD Working Group.Retrospective analysis of 515cases of Crohn’s disease hospitalization in China:nationwide study from 1990to 2003[J].J Gastroenterol Hepatol, 2006,21 (6): 1009-1015.) retrospective study is carried out between IBD inpatient 1990-2003, it collects altogether 3100 UC and 515 CD patients, IBD inpatient in China speculates roughly UC illness rate in trend is gradually increased as the result is shown About 11.6/10 ten thousand, CD is about 1.4/10 ten thousand, and growth pattern is national similar to Japan, South Korea, Singapore etc..
Currently, therapeutic agent mainly includes the following categories for inflammatory bowel disease: (1) aminosalicylic acids preparation, (2) Adrenal Glucocorticoid (GCS), (3) immunosuppressor, (4) monoclonal antibody, (5) antibiotic.But said medicine is deposited In following defect: aminosalicylic acids preparation has larger toxic side effect, such as sulfasalazine (Sulfasalazine, SASP), Its metabolite sulfapryidine can generate adverse reaction；GCS long-time service is also easy to produce adverse reaction；Traditional immunization inhibitor (ratio Such as imuran (AZA), Ismipur (6-MP) and amethopterin (MTX)) it is not effective to all IBD patients, and And adverse reaction is more；Neotype immunosuppressant (such as ciclosporin A (CsA), tacrolimus (FK506) and mould phenol (MMF) etc. It is effective to IBD, but its efficacy and saferry is still up for further evaluating；At present in, severe IBD and high risk patient, if Invalid using first three conventional medicament, then monoclonal antibody inducer remission can be used immediately, but monoclonal antibody price is high It is expensive, and there is also potential risk, such as tuberculosis is suffered from using the patient of IFX or a possibility that histoplasmosis infects It obviously increases, in addition, the illness rate of the diseases such as nervous system Demyelination, congestive heart failure and lymthoma also mentions It is high；Antibiotic can only also take the circumstances into consideration to select, and have the adverse reactions such as Nausea and vomiting, acroparesthesia.
The adverse reactions such as conventional medicament such as aminosalicylic acids preparation, glucocorticoid, immunosuppressor are more, and Dan Ke Grand antibody drug is again expensive, currently, still lacking effective treatment means for inflammatory bowel disease.It is new it is therefore desirable to study The active drug that can treat inflammatory bowel disease of type.
Bacteroides fragilis (bacteroides fragilis) is the member of Bacteroides in Gram-negative anaerobic bacteria, Belong to Bacteroidetes, is totally different from Bifidobacterium, lactic acid bacteria of Firmicutes etc..Bacteroides has 25 strains, is only from The mankind's has 10 strains, and be only from animal has 10 strains, has 5 strains from humans and animals.Bacteroides fragilis is A kind of obligate anaerobes, according to the difference of culture medium and the differences of growth phase, pleomorphism is presented in thalli morphology, under general condition Thallus be rod-shaped, both ends blunt circle, color depth, Neutral colour is shallow and uneven, has pod membrane, without brood cell, unpowered, some have vacuole, Thallus is different in size.According to can synthesize, secrete bacteroides fragilis enterotoxin (BFT) can be classified as produce enterotoxin type fragility intend Bacillus (Enterotoxigenic Bacteroides fragilis, ETBF) and non-production enterotoxin type bacteroides fragilis (NontoxigenicBacteroides fragilis, NTBF).Bacteroides fragilis is as people and animal intestinal tract normal flora A part is primarily present in colon.In addition, respiratory tract, gastrointestinal tract and urogenital mucosa can also be colonized growth.
Summary of the invention
Based on this, the present invention provides a kind of newly answering for bacteroides fragilis (bacteroides fragilis) extract With.
Specific technical solution is as follows:
Application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment inflammatory bowel disease, the fragile quasi- bar Contain bacteroides fragilis capsular polysaccharide A in fungus extract.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~75KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 15KD~65KD；
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 25KD~55KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 35KD~45KD.
In wherein some embodiments, the content of bacteroides fragilis capsular polysaccharide A is in the bacteroides fragilis extract 60-75wt%.
In wherein some embodiments, the bacteroides fragilis is that the fragility that deposit number is CGMCC No.10685 intends bar Bacterium ZY-312.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract will be the following steps are included: (1) will be sent out The first sediment is collected in bacteroides fragilis bacterium solution centrifugation after ferment culture, and first sediment is taken to be added 65-72 DEG C Water adds phenol solution after dissolution, keep 65-72 DEG C of stirring 25-35min, and the first supernatant is collected in centrifugation；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, received Collect aqueous phase solution；
(3) the final concentration of 75-85v/ of dehydrated alcohol to ethyl alcohol is added in the aqueous phase solution being collected into step (2) The second sediment is collected in v%, alcohol precipitation, centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, centrifugation collects second Supernatant, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
In wherein some embodiments, the water that is added in first sediment in step (1), the phenol solution with And the proportion of first sediment is 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is 70-80%.
In wherein some embodiments, step (3) alcohol precipitation be 0-8 DEG C at a temperature of alcohol precipitation 8-16 hours.
In wherein some embodiments, step (4) includes: to take second sediment, adds water to be configured to mass concentration and is The suspension of 8-12% adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, is stirred to react 1.5-2.5 Hour, adjusting pH is 6.5-7.5, and the second supernatant is collected in centrifugation, and desalination of dialysing is freeze-dried to get the bacteroides fragilis Extract.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract further includes the steps that degradation: will Bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the condition of the ultrasound are as follows: 180- 210kHz, 15-25 DEG C.
In wherein some embodiments, the dosage form of the drug includes pill, tablet, granule, capsule, oral solution or pipe Raise preparation.The drug includes people's medication or animal-use drug, can be used for human or animal.The food includes milk powder, cheese, coagulates Cream, yoghourt, ice cream or fermented cereal food.The food can also be animal foodstuff, such as feed etc..The food is also It can be baby food or pet food.
The present invention also provides a kind of bacteroides fragilis extract for preventing and treating inflammatory bowel disease or drugs or food.Tool Body technique scheme is as follows:
A kind of bacteroides fragilis extract or drug or food for preventing and treating inflammatory bowel disease, in the drug or food Containing bacteroides fragilis extract, bacteroides fragilis capsular polysaccharide A is contained in the bacteroides fragilis extract.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~75KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 15KD~65KD；
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 25KD~55KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 35KD~45KD.
In wherein some embodiments, the content of bacteroides fragilis capsular polysaccharide A is in the bacteroides fragilis extract 60-75wt%.
In wherein some embodiments, the bacteroides fragilis is that the fragility that deposit number is CGMCC No.10685 intends bar Bacterium ZY-312.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract the following steps are included:
(1) by the bacteroides fragilis bacterium solution centrifugation after fermented and cultured, the first sediment is collected, takes first precipitating 65-72 DEG C of water is added in object, and phenol solution is added after dissolution, keeps 65-72 DEG C of stirring 25-35min, and centrifugation collects first Supernatant；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, received Collect aqueous phase solution；
(3) the final concentration of 75-85v/ of dehydrated alcohol to ethyl alcohol is added in the aqueous phase solution being collected into step (2) The second sediment is collected in v%, alcohol precipitation, centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, centrifugation collects second Supernatant, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
In wherein some embodiments, the water that is added in first sediment in step (1), the phenol solution with And the proportion of first sediment is 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is 70-80%.
In wherein some embodiments, step (3) alcohol precipitation be 0-8 DEG C at a temperature of alcohol precipitation 8-16 hours.
In wherein some embodiments, step (4) includes: to take second sediment, adds water to be configured to mass concentration and is The suspension of 8-12% adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, is stirred to react 1.5-2.5 Hour, adjusting pH is 6.5-7.5, and the second supernatant is collected in centrifugation, and desalination of dialysing is freeze-dried to get the bacteroides fragilis Extract.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract further includes the steps that degradation: will Bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the condition of the ultrasound are as follows: 180- 210kHz, 15-25 DEG C.
In wherein some embodiments, the dosage form of the drug includes pill, tablet, granule, capsule, oral solution or pipe Raise preparation.The drug includes people's medication or animal-use drug, can be used for human or animal.The bacteroides fragilis extract can be with It is used in combination with other drugs, the other drugs are including but not limited to salicylazosulfapyridine Salicylic Acid Formulations (such as Ai Di Sha, mesalazine), corticosteroid (such as prednisone, dexamethasone) and/or immunosuppressor (such as imuran).The food Product include milk powder, cheese, curdled milk, yoghourt, ice cream or fermented cereal food.The food can also be animal foodstuff, than Such as feed.The food can also be baby food or pet food.
Bacteroides fragilis ZY-312 of the invention is preserved in Chinese microorganism strain preservation management on April 2nd, 2015 Committee's common micro-organisms center (CGMCC), deposit number are CGMCC No.10685, and preservation address is Chaoyang District, Beijing City The institute 3 of North Star West Road 1.
The present inventor accumulates by protracted experience and a large amount of creative experiments researchs, with preparation side of the invention The bacteroides fragilis capsular polysaccharide A that molecular weight is 70KD has unexpectedly been prepared in method, and it was found that the bacteroides fragilis pod membrane Polysaccharide A has the effect of preventing and treating inflammatory bowel disease, and its effect is better than point for extracting and obtaining from bacteroides fragilis NCTC 9343 The capsular polysaccharide A that son amount is 110KD.Further, inventor passes through the bacteroides fragilis capsular polysaccharide A to molecular weight for 70KD Degrade, obtain molecular weight be 5~40KD bacteroides fragilis capsular polysaccharide A, and be surprised to find that molecular weight be 5~ The capsular polysaccharide that it is 110KD than the molecular weight extracted from bacteroides fragilis NCTC 9343 that the capsular polysaccharide A of 40KD, which has, A preferably prevents and treats the effect of inflammatory bowel disease.Prevention and treatment of the bacteroides fragilis capsular polysaccharide A provided by the invention to inflammatory bowel disease Effect is good and is free from side effects to body, while can also be with the Drug combination of other preventing/treating inflammatory bowel disease.This The bacteroides fragilis capsular polysaccharide A that invention provides has edible and prospect in medicine well, provides a kind of suitable people for clinic The non-defective unit for the preventing/treating inflammatory bowel disease that body is taken.
Detailed description of the invention
Fig. 1 is the colony characteristics figure of the bacteroides fragilis ZY-312 of embodiment 1；
Fig. 2 is that the bacteroides fragilis ZY-312 of embodiment 1 carries out the micro- sem observation figure after Gram's staining；
Fig. 3 is the 1H spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 4 is the 13C spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 5 is the COSY spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 6 is the hsqc spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 7 is the HMBC spectrogram that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 8 is the chemical structural formula for the bacteroides fragilis capsular polysaccharide A that embodiment 1 is prepared；
Fig. 9 is daily disease index (DAI) result figure of embodiment 2；
Figure 10 is the colon HE colored graph of each group experiment in embodiment 3；
Figure 11 is the expression feelings of barrier GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 in embodiment 3 Condition figure.
Specific embodiment
The present invention is described in further details below by specific embodiment, these embodiments are only used to illustrate this hair It is bright, it does not limit the scope of the invention.
Bacteroides fragilis used in following embodiment is bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on April 2nd, 2015 (CGMCC), deposit number is CGMCC No.10685, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The preparation of 1 bacteroides fragilis extract of embodiment
(1) fermented and cultured of bacteroides fragilis
By strain streak inoculation in blood plate, Anaerobic culturel 48h.Observe colony morphology characteristic, dyeing property, size, ball Rod-shaped and distribution situation etc..
Colony characteristics: after bacteroides fragilis ZY-312 cultivates 48h on blood plate, round dimpling, translucent, white is presented Color, surface be smooth, not haemolysis, colony diameter between 1-3 mm, referring to Fig. 1.
Form under microscope: bacteroides fragilis ZY-312 carries out gram stain microscopy, is gram-negative bacteria, allusion quotation is presented Rod-shaped, both ends blunt circle and the dense dye of type, not colored part is shaped like vacuole among thallus, referring to fig. 2.
It chooses single bacterium colony to be inoculated in tryptone meat soup progress fermented and cultured 8 hours (temperature is 37 DEG C), gained bacterium Liquid centrifugation, revolving speed 3000r/min are centrifuged 15min, remove supernatant, collect sediment.
(2) preparation of bacteroides fragilis extract
1) bacteroides fragilis bacterium mud (sediment that above-mentioned steps (1) obtain) 200g is taken, 68 DEG C of ultrapure water 750mL are added, After dissolution, adding volume fraction is 75% phenol solution 750mL, is uniformly mixed, and keeps 68 DEG C of stirring extraction 30min, 15000g is centrifuged 20min, takes supernatant liquor.
2) supernatant liquor extracts removal phenol with isometric ether (1.5L), collects supernatant liquor, and repetition is extracted to no benzene Phenol residual.Heating water bath removes ether, collects water phase.
3) water phase 15000g measures volume after being centrifuged 20min, dehydrated alcohol is added, until the final concentration of 80% (volume of ethyl alcohol Score), overnight (12 hours), 15000g is centrifuged 20min to 4 DEG C of alcohol precipitations, takes precipitating.
4) quality of the precipitating in step 3) is weighed, precipitating is configured to mass concentration by the deionized water that certain volume is added It for 10% suspension, is uniformly mixed, the glacial acetic acid aqueous solution that mass concentration is 10% is added, is heated to boiling, persistently stir After mixing reaction 2h, adjusts pH to 7.0,15000g and be centrifuged 20min, collect supernatant.By gained supernatant dialysis desalination, (10KD is saturating Analyse bag), freeze-drying obtains bacteroides fragilis extract.
5) bacteroides fragilis extract described in 30mg step 4) is weighed, 0.5mL D is dissolved in2O, 1 μ l acetone of addition (1H, 2.22；13C, 30.89) calibration.1H, 13C, COSY, HSQC, HMBC are analyzed using 500MHz Bruker nuclear magnetic resonance chemical analyser It composes (Fig. 3-Fig. 7), the bacteroides fragilis extract that confirmation step 4) is collected is capsular polysaccharide A, and purity is about 70%.Pass through GPC (gel permeation chromatography) analysis, the repetitive unit molecular weight of above-mentioned capsular polysaccharide A is 781 as the result is shown, unit repetition number n value It is 89, molecular weight is about 70KD, and molecular formula is-[C31N3O20H47]91, chemical structure is shown in Fig. 8.
(3) preparation of different molecular weight size capsular polysaccharide A
By degrading to the capsular polysaccharide A prepared in (2), the biodegrading process includes but is not limited to the present embodiment Chemical degradation method, physical degradation methods and biological degradation method.For the present embodiment using the method for ultrasound, the ultrasonic method is by pod membrane In 195kHz, 20 DEG C of condition is handled 3 hours, 2 hours, 0.5 hour and 0 hour Polysaccharide A respectively, collects obtain molecular weight respectively Size is the capsular polysaccharide A of 2KD, 5KD, 40KD, 70KD.Use the method for (2) from bacteroides fragilis NCTC 9343 (purchased from beauty State ATCC) in extract obtain molecular weight be 110KD capsular polysaccharide A.
Curative effect of the 2 various dose bacteroides fragilis capsular polysaccharide A of embodiment to the DSS mouse inflammatory bowel disease induced
One, experimental design
The present embodiment induces C57BL/6 mouse enteritis by DSS (dextran sulfate sodium), is then prepared using embodiment 1 Obtained bacteroides fragilis extract (main component is capsular polysaccharide A) treats the C57BL/6 mouse enteritis of induction, examines Survey its therapeutic effect.The present embodiment is by taking molecular weight is the capsular polysaccharide A of 40KD as an example.
Mouse enteritis abductive approach: 60 female C57BL/6 mouse of the present embodiment, every laboratory mice are assigned with one A unique number.Before being grouped to animal, should be marked on the label of mouse cage project number, kind/strain, gender, Cage number and animal number.6 groups, every group 10 are randomly divided into according to the original body mass of mouse using BioBook software.
Start within 0th day, in addition to the 1st group is drunk just common water, the 2nd group to the 6th group mouse drinks the aqueous solution containing DSS, even Continuous to drink 6 days, mouse freely drinks just common water 4 days later (from the 6th day 17:30 to the 9th days afternoon).On the day of starting modeling It is calculated as 0 day.DSS aqueous solution is wrapped up with masking foil, guarantees to be protected from light.The DSS aqueous solution of replacement in every 2 days.In order to guarantee experiment Reliability, the present embodiment adjust the use concentration of DSS according to the average weight and animal state of animal.Substantially principle is as follows:
1.5%DSS: average weight < 17.5g
1.7%DSS:17.5g < average weight < 18g
1.9%DSS:18g < average weight < 18.5g
2.0%DSS:18.5g < average weight < 20g；
Specific experiment grouping and dosage regimen are as shown in table 1:
1 experimental group of table and dosage regimen
Two, observation index and judgement
1, mouse weight
The weight of measurement record animal daily, and the daily routines of animal are observed, recording exceptional situation.
2, daily disease index (DAI) comments grade according to following standard:
Body weight loss (0 ,≤0%；1,1-3%；2,3-6%；3,6-9%；4, > 9%)；
Bloody stool (0, it is negative；2, band bloody stool is just；4, fluid-like bloody stool)；
Stools scored (0, normally；2, loose stools；4, diarrhea)
The score of three parts is added to obtain daily disease index value.
3, serum collection, weight, length and the ulcer area of intestines
9th day, all mouse received to take blood through eye socket after the isoflurane inhalation anesthesia of 3%-5%, separated serum.After blood sampling Mouse receives excess CO2Neck is taken off afterwards to put to death.Abdominal cavity is cut off, mouse Colon and rectum is taken, removes the tissue of pericolonic, measurement is blind from returning Longitudinal length of the valve to anus.Colon and rectum is splitted, is scored content excrement, enteral object is cleaned, integrally takes pictures to colon, is claimed Weight collects colon with spare.
4, statistics and analysis
The average value of the body weight loss of each group mouse, bloody stool, stools scored is calculated separately, the score of three parts is added to obtain Daily disease index value (DAI).Weight, colon lengths are with mean value ± standard false statistic.Using Graphpad Prism, SPSS or Sigmaplot software is for statistical analysis.Specific data are graphically presented.It is poor that P < 0.05 is considered to have statistics It is different.
Three, result and analysis
Use weight, the length of intestines, weight and the ulcer surface of mean value ± standard error (Mean ± S.E.M) statistics animal Product.Intestines correlated variables carries out multiple analysis with ANOVA combination Dunnett ' s.The weight of animals related data is carried out more using ANOVA Compare again.As P < 0.05, then it is assumed that statistically variant.
2 DAI of table scoring
3 Colon and rectum macroscopic evaluation of table
In the present embodiment, positive drug Cyclosporine A (cyclosporine A, CsA) reduces the mouse enteritis of DSS induction Clinical symptoms, including weight loss, three indexs of diarrhea and bloody stool.Cyclosporine A, which is also improved in Colon and rectum, to retain The character of excrement all have statistical difference and compared with model group.
The molecular weight that embodiment 1 provides is that the bacteroides fragilis capsular polysaccharide A high dose group of 40KD is shown significantly Inhibit the drug effect of the mouse enteritis of DSS induction, showing its significantly reduces the increase of Colon and rectum weight caused by enteritis, changes The character of intestinal contents has been apt to it, see Table 3 for details.Compared with model group, the above index all has statistical difference.
In the present embodiment, by comparing the daily disease index (DAI) of 6 groups of experiments it can be found that provided by the invention Daily disease index (DAI) can be effectively reduced in the bacteroides fragilis capsular polysaccharide A of basic, normal, high dosage (see Table 2 for details, Fig. 9).
It is controlled well as it can be seen that bacteroides fragilis capsular polysaccharide A provided by the invention has DSS inducing mouse chordapsus Treatment effect, and any toxic side effect is not shown.
Embodiment 3, intestinal permeability detection
Inflammatory bowel disease seriously destroys intestinal permeability.Intestinal permeability, which is at a normal level, can effectively stop outside The invasion for coming microorganism and toxin are conducive to the interior ambient stable of body, are the guarantees of body health.The present embodiment is to embodiment The bacteroides fragilis capsular polysaccharide A that 1 molecular weight provided is 70KD detects the effect of intestinal permeability, the present embodiment choosing Select the most apparent colonic segment detection following items of the Day9 lesion collected in embodiment 2:
1) histopathologic change: HE dyeing observation tissue change, inflammatory infiltration situation；Transmission electron microscope observing lesion ultra micro knot Structure variation closely connects between observation epithelial cell form, epithelium, epithelial permeability variation.The present embodiment is selected 6 in embodiment 2 The colon that group is collected in Day9 carries out HE dyeing observation according to routine techniques, as a result such as Figure 10.
It is thin with inflammatory it is observed that the epithelium of intestinal mucosa layer integrality of model group mouse is destroyed under microscope Born of the same parents' infiltration, oedema under crypt distorted deformation and mucous membrane.And normally organize control group, ciclosporin A (CsA) and bacteroides fragilis pod membrane The mouse Colon tissue of Polysaccharide A low, middle and high dose groups has no apparent pathological change.
2) intestinal permeability: using everted intestinal sac, and the intestines capsule of preparation is placed in and is added to lactulose and mannitol HBSS buffer (pH7.4), the intracapsular culture solution of intestines after collecting culture, measures through high performance liquid chromatograph.
Specific steps:
(1) preparation of everted intestinal sac: normally organized in Example 2 respectively control group, model group, ciclosporin A (CsA) group, Bacteroides fragilis capsular polysaccharide A (low dosage), bacteroides fragilis capsular polysaccharide A (middle dosage), bacteroides fragilis capsular polysaccharide A The most apparent colonic segment of Day9 lesion, removes mesenterium in (high dose) group, is rinsed well with physiological saline (or buffer), outside Turning over keeps intestinal mucosa outside, and ligation one end forms intestines cryptomere, and perfusion HBSS buffer (pH7.4) ligatures the other end afterwards, and carries out mark Note.
(2) everted intestinal sac prepared by step (1) is placed in the culture solution for being added to lactulose and mannitol, is passed through 95% The mixed gas of oxygen and 5% carbon dioxide cultivates 2h.
(3) culture solution extracted respectively with syringe in intestines capsule carries out high performance liquid chromatograph measurement.
As a result: normally organizing control group, model group, ciclosporin A (CsA) group, bacteroides fragilis capsular polysaccharide A (low dosage) Group, bacteroides fragilis capsular polysaccharide A (middle dosage) group, bacteroides fragilis capsular polysaccharide A (high dose) group intestines capsule in lactulose and The ratio of mannitol content is respectively 0.021 ± 0.006,0.83 ± 0.41,0.029 ± 0.013,0.025 ± 0.009,0.023 ± 0.007 and 0.020 ± 0.008, normal group control group, ciclosporin A (CsA) group, bacteroides fragilis pod are found from testing result Film Polysaccharide A (low dosage) group, bacteroides fragilis capsular polysaccharide A (middle dosage) group, bacteroides fragilis capsular polysaccharide A (high dose) group Being substantially reduced in the odds ratio model group of middle lactulose and mannitol content, significant difference (P < 0.05) have statistics meaning Justice.And normally organize control group, ciclosporin A (CsA) group, bacteroides fragilis capsular polysaccharide A (low dosage) group, bacteroides fragilis pod membrane The ratio of lactulose and mannitol content is close in Polysaccharide A (middle dosage) group, bacteroides fragilis capsular polysaccharide A (high dose) group, Difference is not significant (P > 0.05).As it can be seen that the permeability of mouse Colon section intestinal mucosal barrier obviously increases after DSS is handled, And it can obviously reduce mouse Colon section intestinal mucosal barrier after Cyclosporine A and bacteroides fragilis capsular polysaccharide A treatment Permeability.
3) after intestinal tissue homogenate, TNF-α, IFN-γ, IL-13, IL-17 intestinal wall inflammation: are detected.
Specific step is as follows:
TNF-α, IFN-γ, IL-13, the measurement of IL-17 level cut intestinal segment by kit requirement respectively, remove fatty mesentery Tissue, 10% homogenate of physiological saline preparation on the rocks, -20 DEG C of storages are to be measured.
In addition to blank well, the standard items (100 hole μ L/) of sample or various concentration are added in corresponding aperture respectively, use sealing plate Gummed paper seals reacting hole, and 37 DEG C of insulating boxs are incubated for 90min.
Get rid of liquid in enzyme mark version, board-washing 4 times, every time 5 minutes.
In addition to blank well, it is added in biotin antibody working solution (100 hole μ L/), seals reacting hole, 37 DEG C of perseverances with sealing plate gummed paper Incubation 60min.
With 0.01M PBS board-washing 4 times, every time 5 minutes.
In addition to blank well, Avidin-peroxide complex (ABC) working solution (100 hole μ L/) is added and is sealed with sealing plate gummed paper Firmly reacting hole, 37 DEG C of insulating boxs are incubated for 30min.
With 0.01M PBS board-washing 4 times, every time 5 minutes.
It is added in color developing agent (100 hole μ L/), is protected from light 37 DEG C of insulating boxs and is incubated for 10~15min.
It is added in terminator (100 hole μ L/), measurement OD value at 450nm is engraved in after mixing.
After each sample OD value subtracts the OD value in zero hole, standard curve is established, is calculated in each sample according to standard curve TNF-α, IFN-γ, IL-13, IL-17 content.
The results are shown in Table 4: compared with normally group control group mice, TNF-a, IFN-γ in the homogenate of model group mouse Colon Content obviously increase (P < 0.01), prompt immune imbalance in mouse intestinal mucosa, Pro-inflammatory cytokine levels obviously increase.With Model group mouse is compared, the bacteroides fragilis capsular polysaccharide A that is prepared by Cyclosporine A and embodiment 1 (low dosage, in Dosage and high dose) after treatment, TNF-a in mouse Colon homogenate, IFN-γ content be substantially reduced (P < 0.01), illustrate this The bacteroides fragilis capsular polysaccharide A that invention provides can effectively improve immunologic balance in mouse intestinal mucosa.
TNF-α in the homogenate of 4 colon of table, IFN-γ, IFN-13, IL-17 content
Note: * indicates P < 0.05, significant difference compared with model group.
4) barrier GAP-associated protein GAP mainly has ZO-1, occludin, b-catenin and claudin-1, and the present embodiment use is exempted from Normal group, model group, ciclosporin A (CsA) group and fragility are quasi- in epidemic disease group, Westernblot method detection embodiment 2Day9 The difference of barrier the GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 of bacillus capsular polysaccharide A (high dose).
As a result: the expression of ZO-1 in model group, occludin, b-catenin and claudin-1 barrier GAP-associated protein GAP are obvious Lower than bacteroides fragilis capsular polysaccharide A (high dose) and ciclosporin A (CsA) group, illustrate the bacteroides fragilis prepared through embodiment 1 After capsular polysaccharide A treatment, the expression quantity of barrier GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 obviously increase Add, it is suitable with the expression quantity in Normal group, it is detailed in Figure 11.Illustrate, bacteroides fragilis capsular polysaccharide A provided by the invention can The expression quantity of barrier GAP-associated protein GAP is significantly improved, intestinal mucosal barrier can be effectively adjusted.
Curative effect of the bacteroides fragilis capsular polysaccharide A of 4 different molecular weight of embodiment to inflammatory bowel disease
The present embodiment induces C57BL/6 mouse enteritis by DSS (dextran sulfate sodium dextran sodium sulfate), then The bacteroides fragilis capsular polysaccharide A for 2KD, 5KD, 40KD and the 70KD being prepared respectively using the embodiment of the present invention 1 lures DSS The carry out preventing/treating for the C57BL/6 mouse enteritis led detects the bacteroides fragilis capsular polysaccharide A of different molecular weight to inflammation The therapeutic effect of property enteropathy.The present embodiment is with the bacteroides fragilis capsular polysaccharide A of 2KD, 5KD, 40KD and 70KD of high dose Example.
Referring to embodiment 2 experimental group group technology, by mouse be divided into Normal group, model group, 2KD group, 5KD group, 40KD group, 70KD group and 110KD group.
Dosage regimen is referring to embodiment 2.
Specific experiment grouping and dosage regimen are as follows:
5 experimental group of table and dosage regimen
(1) method of reference implementation example 2 carries out Colon and rectum macroscopic evaluation, and concrete outcome is as follows:
6 Colon and rectum macroscopic evaluation of table
Molecular weight provided by the invention is the bacteroides fragilis capsular polysaccharide A of 5KD, 40KD, 70KD as can be seen from the above table The drug effect for showing the significant mouse enteritis chordapsus for inhibiting DSS induction, is in particular in that its reduces inflammation significantly The increase of Colon and rectum weight caused by disease property enteropathy, improves the character of intestinal contents, see Table 6 for details.With 2KD group and 110KD group It comparing, 5KD, 40KD, 70KD are reducing Colon and rectum weight caused by inflammatory bowel disease and stool scores show better activity, P < 0.05, statistical difference is significant.
(2) method of reference implementation example 3 detects intestinal permeability, normally organizes control group, model group, 2KD, 5KD group, 40KD Group, 70KD group, the ratio of lactulose and mannitol content is respectively 0.023 ± 0.007,0.84 ± 0.52,0.76 in 110KD group ± 0.42,0.025 ± 0.014,0.023 ± 0.004,0.024 ± 0.008 and 0.120 ± 0.014.Just from testing result discovery Often group control group, 5KD group, 40KD group, 70KD group, lactulose and the odds ratio 2KD group of mannitol content, model in 110KD group Being substantially reduced in group, difference is extremely significant (P < 0.01), has statistical significance.In addition, compared with 110KD group, 5KD group, The ratio of lactulose and mannitol content is substantially reduced in 40KD group and 70KD group, significant difference (P < 0.05), has statistics Meaning.
Through this embodiment as can be seen that the present invention reduces its point by degrading to bacteroides fragilis capsular polysaccharide A Son amount and viscosity, can be enhanced its bioactivity, be especially to have in the section 5KD -70KD to inflammatory bowel disease in molecular weight Better curative effect.
Curative effect of the 5 different strains bacteroides fragilis capsular polysaccharide A of embodiment to inflammatory bowel disease
The ultrasonic method that the present embodiment is recorded using embodiment 1 degrades to the capsular polysaccharide A that molecular weight is 110KD (ultrasound condition: 195kHz, 25 DEG C, 0.5 hour), and the capsular polysaccharide A that molecular weight is 70KD is collected, it is denoted as NCTC 9343- 70KD group, and the capsular polysaccharide A (being denoted as ZY-312-70KD group) for being 70KD with the molecular weight extracted from ZY-312 is carried out Comparison, evaluates its curative effect to inflammatory bowel disease.It is macro to detect Colon and rectum respectively for the method that the present embodiment reference implementation example 4 is recorded It sees evaluation and intestinal permeability, concrete outcome is as follows:
7 Colon and rectum macroscopic evaluation of table
In ZY-312-70KD group, NCTC 9343-70KD group the ratio of lactulose and mannitol content be respectively 0.023 ± 0.007、0.025±0.014。
It can be seen from the results above that obtaining the capsular polysaccharide that molecular weight is 110KD for extracting from 9343 bacterial strain of NCTC A degrades, and may be implemented with the capsular polysaccharide A from the extraction of ZY-312 bacterial strain to therapeutic effect similar in inflammatory bowel disease.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (10)

1. application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment inflammatory bowel disease, which is characterized in that institute It states and contains bacteroides fragilis capsular polysaccharide A in bacteroides fragilis extract.
2. application according to claim 1, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A be 5~ 75KD。
3. application according to claim 1, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A is 35KD~45KD.
4. application according to claim 1-3, which is characterized in that the bacteroides fragilis is that deposit number is The bacteroides fragilis ZY-312 of CGMCC No.10685.
5. application according to claim 1-3, which is characterized in that the preparation side of the bacteroides fragilis extract Method the following steps are included:
(1) by the bacteroides fragilis bacterium solution centrifugation after fermented and cultured, the first sediment is collected, first sediment is taken to add Enter 65-72 DEG C of water, phenol solution is added after dissolution, keep 65-72 DEG C of stirring 25-35min, the first supernatant is collected in centrifugation Liquid；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, collect water Phase solution；
(3) final concentration of 75-85v/v% of the dehydrated alcohol to ethyl alcohol, alcohol are added in the aqueous phase solution being collected into step (2) Heavy, the second sediment is collected in centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, the second supernatant is collected in centrifugation Liquid, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
6. application according to claim 5, which is characterized in that the water that is added in first sediment in step (1), The phenol solution and the proportion of first sediment are 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is 70-80%.
7. application according to claim 5, which is characterized in that step (3) alcohol precipitation is the at a temperature of alcohol precipitation at 0-8 DEG C 8-16 hours.
8. application according to claim 5, which is characterized in that step (4) includes: to take second sediment, and water is added to match The suspension that mass concentration is 8-12% is made, adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, stir Mix reaction 1.5-2.5 hour, adjustings pH be 6.5-7.5, centrifugation, collect the second supernatant, dialysis desalination, be freeze-dried to get The bacteroides fragilis extract.
9. application according to claim 5, which is characterized in that the preparation method of the bacteroides fragilis extract further includes The step of degradation: bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the ultrasound Condition are as follows: 180-210kHz, 15-25 DEG C.
10. a kind of bacteroides fragilis extract for preventing and treating inflammatory bowel disease or drug or food, which is characterized in that the medicine Contain bacteroides fragilis extract in object or food, contains bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.
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